1、BRITISH STANDARD BS EN 1141:1995 Method for Determination of proline content of fruit and vegetable juices: Spectrometric method The European Standard EN1141:1994 has the status of a British Standard UDC663.81/.82:620.1:543.42:547.747BSEN 1141:1995 This British Standard, having been prepared under t
2、he directionof the Consumer Products and Services Sector Board, was published under theauthority of the Standards Board and comes intoeffect on 15 May 1995 BSI03-2000 The following BSI references relate to the work on this standard: Committee reference AW/21 Draft for comment90/51496 DC ISBN 0 580 2
3、3168 2 Cooperating organizations The European Committee for Standardization (CEN), under whose supervision this European Standard was prepared, comprises the national standards organizations of the following countries: Austria Oesterreichisches Normungsinstitut Belgium Institut belge de normalisatio
4、n Denmark Dansk Standard Finland Suomen Standardisoimisliito, r.y. France Association franaise de normalisation Germany Deutsches Institut fr Normung e.V. Greece Hellenic Organization for Standardization Iceland Technological Institute of Iceland Ireland National Standards Authority of Ireland Italy
5、 Ente Nazionale Italiano di Unificazione Luxembourg Inspection du Travail et des Mines Netherlands Nederlands Normalisatie-instituut Norway Norges Standardiseringsforbund Portugal Instituto Portugus da Qualidade Spain Asociacin Espaola de Normalizacin y Certificacin Sweden Standardiseringskommission
6、en i Sverige Switzerland Association suisse de normalisation United Kingdom British Standards Institution Amendments issued since publication Amd. No. Date CommentsBSEN 1141:1995 BSI 03-2000 i Contents Page Cooperating organizations Inside front cover National foreword ii Foreword 2 1 Scope 3 2 Norm
7、ative references 3 3 Symbols 3 4 Principle 3 5 Reagents 3 6 Apparatus 3 7 Procedure 3 8 Calculation 4 9 Precision 4 10 Test report 4 Annex A (informative) Bibliography 6 Annex B (informative) Statistical results of inter-laboratory test 6 National annex NA (informative) Committees responsible Inside
8、 back cover National annex NB (informative) Cross-references Inside back cover Table B.1 6BSEN 1141:1995 ii BSI 03-2000 National foreword This British Standard has been prepared under the direction of the Consumer Products and Services Sector Board and is the English language version of EN1141:1994
9、Fruit and vegetable juices Spectrometric determination of proline content, published by the European Committee for Standardization (CEN). EN1141 was produced as a result of international discussions in which the United Kingdom took an active part. A British Standard does not purport to include all t
10、he necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pagesi andii,
11、the EN title page, pages2 to6, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover.EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN1141 October
12、1994 UDC663.81/.82:620.1:543.42:547.747 Descriptors: Food products, beverages, fruit and vegetable juices, chemical analysis, determination of content, proline, spectrophotometric analysis English version Fruit and vegetable juices Spectrometric determination of proline content Jus de fruits et de l
13、gumes Dosage de la proline par spectromtrie Frucht und Gemsesfte Spektralphotometrische Bestimmung des Prolingehalts This European Standard was approved by CEN on1994-09-29. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this Europ
14、ean Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English,
15、 French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Denmark, Finla
16、nd, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN European Committee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart 36, B-1050 Bru
17、ssels 1994 Copyright reserved to CEN members Ref No. EN1141:1994 EEN 1141:1994 BSI 03-2000 2 Foreword This European Standard has been prepared by the Technical Committee CEN/TC174, Fruit and vegetable juices Methods of analysis, the secretariat of which is held by AFNOR. This European Standard shall
18、 be given the status of a National Standard, either by publication of an identical text or by endorsement, at the latest by April1995, and conflicting national standards shall be withdrawn at the latest by April1995. Annexes designated “informative” are given only for information. In this standard A
19、nnex A and Annex B are informative. According to the CEN/CENELEC Internal Regulations, the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden
20、, Switzerland and United Kingdom.EN 1141:1994 BSI 03-2000 3 1 Scope This European standard specifies a spectrometric method for the determination of proline in fruit and vegetable juices and related products. 2 Normative references This European Standard incorporates by dated or undated reference, p
21、rovisions from other publications. These normative references are cited at the appropriate places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this European Standard only when incorporated in
22、it by amendment or revision. For undated references the latest edition of the publication referred to applies. ISO5725:1986, Precision of test methods Determination of repeatability and reproducibility for a standard test method by inter-laboratory tests. ISO3696:1987, Water for analytical laborator
23、y use Specification and test methods. 3 Symbols For the purposes of this standard, the following symbols apply: 4 Principle Proline forms a coloured complex with ninhydrin. This is extracted with n-butyl acetate and the absorbance at509nm of the coloured extract is measured spectrometrically. 5 Reag
24、ents 5.1 General. Use only reagents of recognized analytical grade and only water in accordance with at least grade3 of ISO3696:1987. 5.2 Ninhydrin solution in ethyleneglycol monmethyl ether, (Ninhydrin)=30g/l. Prepare once a week and store in a dark container at4 C 2 C 5.3 n-Butyl acetate 5.4 Formi
25、c acid, (CH 2 O 2 )=98% (m/m) 1% (m/m). 5.5 Sodium sulfate, (anhydrous). 5.6 L-Proline 6 Apparatus Usual laboratory apparatus and, in particular, the following: 6.1 Glass cuvettes, of10mm optical path length, and which do not have significant absorption at509nm. 6.2 Spectral-line photometer, with me
26、rcury lamp and filters for measuring at509nm. 6.3 Spectrometer, (variable wavelength) for measuring at509nm (alternative to6.2). 6.4 Test tubes, stoppered, approximately25ml capacity. The stoppered test tubes used shall have similar wall thickness. 6.5 Funnels, approximately65mm diameter. 6.6 Filter
27、 papers, hydrophobic silicone coated, diameter110mm. 6.7 Laboratory timer 6.8 Water bath, temperature control 2 C. 7 Procedure 7.1 Preparation of the test sample Normally products shall not be pretreated and their analysis by this method shall be on a volumetric basis, results being expressed per li
28、tre of sample. The analysis of concentrated products may also be carried out on a volumetric basis, after dilution to a known relative density. In this case, the density shall be indicated. Based on a weighed sample and taking the dilution factor for analysis into account, the results may also be ex
29、pressed per kilogram of product. In products with high viscosity and/or very high content of cells (for example pulp), determination on the basis of a weighed test sample is the usual procedure. Samples with proline content50mg/l and over should be diluted with water as follows: Strongly coloured pr
30、oducts with less than50mg L-proline should be diluted1 part sample+1 part water or1 part sample+4 parts water according to their colour intensity. 7.2 Measurement of extinction coefficient Dissolve L-proline in water to give a stock solution, r (L-proline)=100mg/l. Dilute this stock solution to give
31、 solutions with L-proline concentrations=5mg/l,10mg/l,25mg/l, 40mg/l and50mg/l. Treat1,0ml portions of these solutions as described in Section7.3 below. c Substance concentration; Mass concentration; Mass fraction. 50mg/l 499mg/l dilute1 part sample+9 parts water; 500mg/l 1000mg/l dilute1 part sampl
32、e+19 parts water.EN 1141:1994 4 BSI 03-2000 Plot the absorbance values obtained against L-proline content (mg/l). This should give a straight line passing through the origin. Since the extinction is subject to variation, it should be determined with each set of samples as follows: Make three separat
33、e measurements of the absorbance at different points on the standard curve (e.g.10mg/l,25mg/l and40mg/l). Calculate the extinction coefficient () using the equation: The average extinction coefficient is calculated from three closely agreeing values. 7.3 Ninhydrin reaction and development of colour
34、Pipette1,0ml of ether test sample or standard solution (see7.2) into a stoppered test-tube, followed by1ml formic acid(5.4) and2ml of ninhydrin solution(5.2). Mix the contents well after adding each reagent. Then place the test-tubes in a boiling water bath in which the water level is higher than th
35、e level of the solutions in the tubes. The boiling water bath shall not be replaced by any other source of heat (e.g.Thermoblock). Ensure that the water bath remains boiling. Leave the tubes in the boiling water bath for exactly15min. Then cool the tubes for5min to10min in water at about20 C. Add10m
36、l of n-butyl acetate to each tube, insert the stopper and shake thoroughly to extract the colour into the organic phase. Pour the whole solution into a folded hydrophobic filter paper(6.6) containing2g to3g of anhydrous sodium sulfate and collect the filtrate. After allowing to stand for about15min
37、the absorbance of the organic phase (filtrate) is measured in a10mm cuvette at509nm against a blank solution prepared in the same way from1ml of sample solution but using only ethyleneglycol monomethyl ether instead of the ninhydrin solution, all other conditions remaining as described above. 8 Calc
38、ulation Calculate the proline content, (Proline) using the value of the extinction coefficient determined in7.2 and report in milligrams per litre, without a decimal place. (Proline)= A Take into account the dilution factor and the relation of the value to mass or volume. If a concentrated product h
39、as been diluted to single strength, report the relative density of the single strength sample. 9 Precision Details of the inter-laboratory test on the precision of the method are summarized in Annex B. The values derived from the inter-laboratory test may not be applicable to analyte concentration r
40、anges and matrices other than given in Annex B. 9.1 Repeatability The absolute difference between two single results found on identical test material by one operator using the same apparatus within the shortest feasible time interval will exceed the repeatability value r in not more than5% of the ca
41、ses. The values are: 9.2 Reproducibility The absolute difference between single test results on identical test material reported by two laboratories will exceed the reproducibility value R in not more than5% of the cases. The values are: 10 Test report The test report shall contain the following dat
42、a: all information necessary for the identification of the sample (kind of sample, origin of sample, designation); a reference to this European Standard; the date and type of sampling procedure (if possible); where: s is the proline concentration of the standard solution, in mg/l; A is the correspon
43、ding absorbance at that concentration. s A - = where k 50mg/l r=2,0mg/l; 50mg/l k 500mg/l r=11mg/l; 500mg/l k 1000mg/l r =23mg/l. where is the measured content, calculated as mean value from the two single test results. k 50mg/l R =0,8mg/l. 50mg/l k 500mg/l R=5,5mg/l 500mg/l k 1000mg/l r =3,5mg/l. w
44、here is the measured content, calculated as the mean value from the two single test results.EN 1141:1994 BSI 03-2000 5 the date of receipt; the date of test; the test results and units in which they have been expressed; whether the repeatability has been verified; any particular points observed in t
45、he course of the test; any operations not specified in the method or regarded as optional, which might have affected the results.EN 1141:1994 6 BSI 03-2000 Annex A (informative) Bibliography 1 Determination of proline: No49,1983. In:AnalysesCollection/International Federation ofFruit Juice Producers
46、. Loose-leaf edition, as of1989. Zug: Swiss Fruit Union. 2 S.Wallrauch, Flssiges Obst, 1976, vol.43, p430-437 Prolinbestimmung in Fruchtsften, Bedeutung fr die Beurteilung. (Determination of proline in fruit juices, importance in (authenticity) judgements.). 3 Untersuchung von Lebensmitteln: Bestimm
47、ung des Prolingehaltes in Fruchtsften: L31.00-7,1980-05 Food analysis: Determination of proline content in fruit juices: L31.00-7,1980-05 In: Amtliche Sammlung von Untersuchungsverfahren nach 35 LMBG; Verfahren zur Probenahme und Untersuchung von Lebensmitteln, Tabakerzeugnissen, kosmetischen Mittel
48、n und Bedarfsgegenstnden/Bundesgesundheitsamt In: Collection of official methods under article35 of the German Federal Foods Act: Methods of sampling and analysis of foods, tobacco products cosmetics and commodity goods/Federal Health Office Loseblattausgabe; Stand31.12.1991, Bd.1. Loose-leaf editio
49、n, as of1991-12-31, Vol.1. Berlin, Kln: Beuth Verlag GmbH. Annex B (informative) Statistical results of the inter-laboratory test In accordance with ISO5725:1986, the following parameters have been defined in an inter-laboratory test. (For literature pertaining to the method see Annex A). The test was conducted by the Max von Pettenkofer Institute of the Federal Health Office, Food Chemistry Department, Berlin, BRD. Table B.1 Year of inter-lab
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